ministry of agriculture fisheries and food csg 15...

CSG 15 (Rev. 12/99) 1

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development

Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title Development of a routine system for Agrobacterium-mediated transformation of barley

MAFF project code CEO 159

Contractor organisation and location

The Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft, Leicester, LE7 9SU

Total MAFF project costs £ 150,143

Project start date 01/12/98 Project end date 30/09/01

Executive summary (maximum 2 sides A4)

This project was funded in response to the identification of an urgent need for a routine, high frequency, barley transformation system to facilitate both fundamental research and crop improvement. The barley transformation systems which were available in 1998, tended to be based on the biolistic method which was seem to have many shortfalls including dependence on high-cost instrumentation, complex re-arrangement of multiple copies of transgenes, and high frequency of transgene silencing in the transgenic plants which were produced. Agrobacterium-mediated DNA delivery promised to be an attractive alternative, which, when optimised, would permit facile, routine, low cost, high frequency transformation of barley. Outcomes of the project were: (a) highly efficient regeneration systems were established for immature embryos of barley cultivars Golden

Promise and Dissa and microspores of barley cultivars Dissa and Igri. Embryogenic callus was produced from immature embryos 14 days after anthesis and microspores at mid- late uninucleate stage on modified MS (Cho et al., 1998) and N6 (Wu et al., 1998) medium respectively.

(b) efficient binary vector systems were defined in close collaboration with scientists at The John Innes Centre

and the procedures for growth of highly potent Agrobacterium strains were refined. Co-cultivation procedures were optimised by assessing the effect of wounding, vir gene induction and inoculation densities on the transformation frequency. It was found that EHA101/pBECKS400 or EHA101/pBECKS2000, a super virulent Agrobacterium strain carrying normal binary vectors; LBA4404/pTOK233, an ordinary strain carrying a super virulent vector; and AGL1/pAL155 + 156, a super virulent strain carrying vectors supplemented with additional vir genes, were the most potent combinations. They delivered T-DNA at high

Project title

Development of a routine system for Agrobacterium-mediated transformation of barley

MAFF project code

CEO 159

CSG 15 (1/00) 2

frequency (as indicated by the GUS assay). LBA4404/pBECKS400 or LBA4404/pBECKS2000 were not effective in barley transformation. One tenth of the normal MS salt concentration in the inoculation and co-cultivation medium and an Agrobacterium inoculum density of 1-4 at OD600 proved to be more efficient for transformation than other treatments. The inclusion of acetosyringone and application of extra wounding proved to be unnecessary.

(c) parameters and procedures for selection of stably transformed barley were established. Selection for stable

transformed callus was carried out for 2 X 3-4 week cycles, on callus induction medium containing hygromycin (50 mg/l). Plant regeneration/selection was carried out on FHG medium supplemented with hygromycin (25 mg/l). Transformation frequencies of 0.3 – 3% were achieved.

Flow charts for Agrobacterium-mediated transformation of barley embryogenic calluses derived from immature embryos (Figure A) and microspores (Figure B) are shown below.

Figure A

Isolation of immature embryos 14 days after anthesis

? Induction of embryogenic callus on modified MS medium (Cho et al., 1998) supplemented with 2.5 mg/l dicamba

? Cocultivation with Agrobacterium EHA101 pBECKS 2000 (OD600 value of about 1.0 ) for 2-4 days at 25 ºC

? Disinfection with cefotaxime (400 mg/l) and culture on modified MS medium (Cho et al., 1998) containing cefotaxime for 1-2

weeks ?

Callus selection for 2 X 3-4 week cycles on callus induction medium (Cho et al., 1998) containing hygromycin (50 mg/l) ?

Plant regeneration/selection on FHG medium (Hunter, 1998) containing hygromycin (25 mg/l) ?

PCR and Southern blot analysis of putative transformants ?

Genetic analysis of progeny of the transformants Figure B

Isolation of microspores at mid-late uninucleate stage ?

Induction of embryogenic callus on modified N6 medium (Wu et al., 1998) supplemented with 0.5 g/l 2,4D plus 0.5 mg/l kinetin ?

Cocultivation with Agrobacterium EHA101 pBECKS 2000 (OD600 value of about 1.0 ) for 2-4 days at 25 ºC ?

Disinfection with cefotaxime (400 mg/l) and culture on modified MS medium (Cho et al., 1998) containing cefotaxime for 1-2 weeks

? Callus selection for 2 X 3-4 week cycles on callus induction medium (Cho et al., 1998) containing PPT (5 mg/l)

? Plant regeneration selection on FHG medium (Hunter, 1998) containing PPT (5 mg/l)

? PCR and Southern blot analysis of putative transformants

? Genetic analysis of progeny of the transformants

The genetic analyses of plants produced via these protocols have not been completed. Further molecular and genetic analyses of plants emerging from the on-going production processes will be carried out by The Norman Borlaug Institute team at the Institute’s own expense and the results will be submitted to the DEFRA.

Project title


MAFF project code

CEO 159

CSG 15 (1/00) 3

The new transformation systems will facilitate advances in fundamental molecular genetics and barley crop improvement and will also have relevance to correspond ing research in other cereals and grasses.

Project title


MAFF project code

CEO 159

CSG 15 (1/00) 4

Scientific report (maximum 20 sides A4) SCIENTIFIC OBJECTIVES AND PRIMARY MILESTONES Scientific objectives a) To establish regenerable barley cultures as targets for inoculation by Agrobacterium including:

i) the establishment of immature embryo cultures ii) the establishment of microspore and anther cultures iii) the establishment of cell suspens ion cultures from a) i) and a ii)

b) To establish an effective inoculation procedure by: i) determining the optimum growth conditions and inoculation densities of various Agrobacterium

strains ii) using established vectors and investigating the effects of virulence induction by virG mutants and vir

gene inducers on transformation frequency iii) assaying the effect of wounding of target materials on transformation frequency

c) To select and screen transgenic plants by applying the established and optimized transformation system in large scale experiments: i) to apply the transformation system to young immature embryo materials on a large scale; ii) to apply the transformation system to cell cultures derived from immature embryos or microspores; iii) to fine-tune the transformation system during the implementation of c) i) and c) ii);

d) To undertake molecular and genetic analysis of transgenic plants and their progeny. Primary milestones

Milestones Target date Title a) i) 01/5/99 Establish highly regenerable

cultures from dissected immature embryo

a) ii) 01/5/99 Establish highly regenerable cultures from microspores or anthers

a) iii) 01/5/99 Establish cell suspension cultures from a) i) and a ii)

b) i) 01/10/99 Test growth conditions and inoculation densities for Agrobacterium strains

b) ii) 01/12/99 Define the earliest time and the best length for Agrobacterium inoculation of each culture

b) iii) 01/12/99 Assess the effect of vir gene inducers on transformation frequency

b) iv) 01/12/99 Assess the effect wounding of target materials on transformation frequency

c) 01/5 2001 Large scale experiment to

Project title


MAFF project code

CEO 159

5

produce transformed barley plants

c) i) 01/5 2001 Large scale transformation of immature embryos of barley

c) ii) 01/5 2001 Large scale transformation of barley cell cultures

c) iii) 01/5 2001 Fine-tuning of the established transformation system

d) 30/9/ 2001 Molecular and genetic analysis of the transformed plants

RESULTS a) Establishment of regenerable barley cultures Procedures for in vitro culture of barley immature embryos (IEs) to give an intermediate callus phase followed by plant regeneration, have been developed and used in transformation experiments exploiting the biolistic approach (Wan and Lemaux, 1994; Cho et al., 1998). The tissue culture protocols established by Cho and co-workers (1998) were exploited in the work reported here. IEs of barley cultivar Golden Promise were used directly for Agrobacterium-mediated transformation (See Section 2). Efficient transient gene expression was demonstrated but the recovery of stably transformed plants proved problematic. This necessitated a quest for other approaches including the development of regeneration systems based on embryogenic callus cultures and cell suspension cultures. Regenerable cultures were established from IEs of the barley cultivars Golden Promise and Dissa. IEs were isolated 14 days after anthesis and cultured on standard MS media (Murashige and Skoog, 1962) for inducing callus. Initially, the plant growth regulator used was 2,4-D at a concentration of 2.5 mg/l. Subsequently 2,4-D was replaced by dicamba (2.5 mg/l) and MS medium was replaced by a modified MS medium (Cho et al., 1998). It was found that dicamba induced more embryogenic cultures than 2,4-D and nearly doubled the numbers of embryogenic calluses (Table 1). This increase was expected to have an impact on the outcome of transformation by improving the regenerability of selected transformed cultures. Regenerable cultures have also been established from microspores of the barley cultivars Dissa and Igri. Microspores (at mid- late uninucleate stage) were cultured on modified N6 medium (Wu et al., 1998) using 2,4-D (0.5 mg/l) and kinetin (0.5 mg/l) as the plant growth regulators. Three weeks were required for producion of embryogenic callus. Table 1. Effect of 2,4-D and dicamba on barley callus induction frequencies (%) and embryogenic callus production (+)

Cultivar 2,4-D (2.5 mg/l) Dicamba (2.5 mg/l) Golden Promise 76 %, ++ 85%, +++ Dissa 82%, ++ 88%, ++++

Cell suspension cultures were established by transferring IE-derived or microspore-derived calluses to liquid MS medium supplemented with 2 mg/l 2,4-D. After weekly subculture for two months, fine suspensions were obtained.

Project title


MAFF project code

CEO 159

6

b) Establishment of an effective Agrobacterium inoculation procedure To study T-DNA transfer from Agrobacterium to barley cells a novel series of vectors, pBECKS400, has been constructed (Figure 1). This set of binary vectors incorporates various visual reporters (maize C1/Lc, E. coli gusA and the synthetic jellyfish (Aequoria victoria) green fluorescent protein gene sgfp-S65T) and selectable marker genes (nptII, bar and hpt) within the T-DNA (McCormac et al., 1998).

Figure 1. The structure of the T-DNA region of binary vectors from the pBECKS400 series. Intact IEs from barley and wheat were inoculated with Agrobacterium EHA101 strain harbouring different pBECKS400 vectors. All the marker genes used were equally useful for detection of early transformation events. However, as high levels of expression of C1/Lc or gfp markers are sometimes associated with deleterious effects on the growth of transformants (Quattrochio et al., 1993; Chiu et al., 1996), gusA gene was chosen for routine use. All the experiments concerning optimization of transformation parameters were carried out with immature barley embryos as the targets. A.tumefaciens AGL1/pAL156 + 157 strain/vector combination was chosen on the basis of the data that described the impact of strain/vector “virulence” on the Agrobacterium transformation efficiency in cereals (Hiei et al., 1994). The importance of promoter (Christensen et al., 1992: Chibbar et al., 1993; Corneyo et al., 1993; Taylor et al., 1993; Weeks et al., 1993; Wan and Lemaux, 1994) and the type of marker gene used ( Pang et al., 1996; McCormac et al., 1998) for assessment of the effectiveness of delivery procedures at an early pre- integration state were also considered. A detailed description of the system is provided below (see Section 2.3.1). Impact of medium composition The importance of medium composition for achieving a high efficiency of Agrobacterium-mediated transformation in cereals has been pointed out by several authors ( Li et al., 1992; Cheng et al., 1997; Zhang et

Project title


MAFF project code

CEO 159

7

al., 1997). The optimal combinations of basal salt strength, 2,4-D and acetosyringone were investigated for IEs of barley (Table 2). Table 2. Impact of medium composition on the frequency and distribution of Agrobacterium-mediated T-DNA transformation of barley embryos as indicated by GUS histochemical assay. Freshly isolated IEs of barley were inoculated with A. tumefaciens (OD600 = 1.5) for 30 min and then co-cultivated for 3 days. Media used are as follows: MSO (MS salts and vitamins + 30 g/l maltose + 1g/l casein hydrolysate (CH), pH 5.8; MS1 (MSO + 2.5 mg/l 2,4-D); MS2 (MS0 + 100 uM acetosyringone (AS); MS3 (MS0 + 2.5 mg/l 2,4-D + 100 uM AS); RMSO (0.1 MS salts + MS vitamins + 30 g/l maltose + 1 g/l CH, pH 5.8); RMS1 (RMS0 + 2.5 mg/l 2,4-D); RMS2 (RMS0 + 100 uM AS); RMS3 (RMSO + 2.5 mg/l 2,4-D + 1000 uM AS). Values are the means + standard deviations of four independent experiments.

Medium MS0 MS1 MS2 MS3 RMSO RMS1 RMS2 RMS3 % embryos with blue spots

78 + 4.0 67 + 3.9 82 + 3.6 88 + 3.6 100 + 2.5 96 + 1.8 100 + 0 100 + 0

Av No of blue spots/embryo

6.7 + 0.3 1.7 + 0.3 8.4 + 0.6 6.8 + 0.4 76.8 + 6.4 30.2 + 3.7 55.4 + 5.9 22.6 + 3.1

Av No blue spots/groove area

4.8 + 0.5 1.6 + 0.2 5.6 + 0.4 4.2 + 0.4 23.3 + 2.9 8.2 + 1.0 13.1 + 2.5 7.1 + 1.0

Av No blue spots /scutellum

1.1 + 0.1 0.1 + 0.0 0.7 + 0.2 1.6 + 0.2 45.1 + 6.7 19.9 + 3.2 31.6 + 5.0 13.3 + 2.1

Decreasing the MS basal salts level to 1/10 of standard strength led to a ten-fold increase in the total number of GUS foci in barley IEs. Furthermore, the distribution of the gusA expressing cells was clearly altered - shifting from the adaxial/groove area, which has poor regeneration capacity to the abaxial/scutellar surface (Figure 2). The overall frequency of GUS foci and their relative distribution over each embryo was also altered when AS was supplied to the MS 1 X medium at a final concentration of 100 ?M. The effect was more pronounced in combination with 0.1X MS. Medium with 1/10 salt strength supplemented with 1000 ?M AS did not give a further increase of transformation frequency, but had the opposite effect. Exclusion of 2,4-D from inoculation and co-cultivation treatments led to an increase in the overall frequency of transformation events in barley IEs. Cheng et al. (1997) reported that the use of Pluronic F-68 (surfactant) brought about an enhanced interaction of the Agrobacterium vectors with wheat IEs. Experiments with barley however showed that supplementation of the medium with the agent at final concentrations of 0.02%, 0.05%, 0.2% or 0.5% had a negative impact on transformation efficiency. Glucose, added at a concentration of 5, 10 or 20 g/l had the same effect (data not shown). The importance of the gelling agent used is outlined in Table 3. Phytagel (2.5 g/l, Sigma) was found to be associated with a markedly lower overall frequency of T-DNA transfer events as compared to agar powder (8 g/l, LAB) or agarose (6 g/l, FMC Bioproducts) . Of these last two the agarose product was judged qualitatively to be associated with a slightly higher and more consistent level of gusA expression, and hence, was used as standard throughout the subsequent experiments. Table 3. Impact of medium gelling agent on efficiency of Agrobacterium-mediated transformation of barley embryos. Freshly isolated IEs were inoculated with A. tumefaciens (OD600 = 1.5) for 30 min in MS3 medium and then co-cultivated for 3 days on MS3 medium solidified with different agents. GUS foci were then scored and compared. The values are means with standard deviations indicated, of five independent experiments.

Gelling agent/concentration. Phytagel/ 2.5 g/l Agar powder/8 g/l Agarose/6 g/l % embryos with GUS foci 58 + 5.2 77 + 3.7 90 + 2.8 Av No GUS foci/embryo 2.7 + 0.3 5.1 + 0.3 6.5 + 0.4 Av No GUS foci/groove region 2.0 + 0.3 3.8 + 0.3 4.1 + 0.4 Av No GUS foci/scutellum 0.1 + 0.0 0.7 + 0.1 1.1 + 0.1

Project title


MAFF project code

CEO 159

8

Figure 2. GUS expression patterns in barley IEs (c – g: Golden Promise; h – Dissa) after inoculation with A. tumefaciens AGL1/pAL156 + 157; (a – b) – labelled diagrams of the different regions of a barley IE: a – abaxial scutellum side; bsr – abaxial scutellum region; gr – groove region; b – adaxial scutellum side; dsr – adaxial scutellum region; GUS staining: c – after 30 min inoculation and 3 days co-cultivation on MS3; e – after 8 h inoculation and 3 days co-cultivation on RMS3; f,g – precultured for 2 days on RMS3 embryos after 24 h inoculation nd 2 days co-cultivation on RMS3; h – after 2 h inoculation and 3 days co-cultivation on RMS3; d – on developing roots. Impact of inoculum density and duration of inoculation The density of the Agrobacterium suspension had a strong impact on the transformation outcomes. An increase of the OD 600 of the bacterial inoculum from 0.1 to 4.0 caused a directly proportional effect on transformation frequency. This increase in the number of GUS foci was accompanied by a shift of the targeting to the abaxial scutellar surface. The effect was equally valid for both 0.1 X and 1 X MS salt concentration in the medium. The importance of duration of the inoculation period (based on OD 600 = 4.0) was also examined. When the bacterial suspension was applied to freshly isolated IEs on medium containing 0.1 X the standard salt strength, extension of the inoculation period from 0.5 h to 8 h produced a slight increase in the levels of gusA expression with a slight shift of transformation events to the scutellum region. A 24 h period of inoculation produced no observable gusA expression at any site and the embryos usually died. But if they were pre-cultured for 2 days on 0.1 X salt strength medium, and then subjected to 24 - 48 h inoculation, a large number of GUS foci in the scutellum region appeared (Figure 2f).

Project title


MAFF project code

CEO 159

9

Impact of embryo wounding Neither electroporation nor axis dissection produced an increase of transformation efficiency. Only in barley cultivar Dissa was enhanced transformation observed in or very near areas that were wounded by forceps during the explant preparation process. In conclusion three key inoculation and co-cultivation parameters have been identified that tend to enhance transformation frequency through an overall increase of T-DNA transfer and a shift of transformation events towards regenerable tissues. These factors are: 1. the MS basal salt concentration, 2. the type of gelling agent in the medium and 3. the density of the bacterial suspension. 1/10 of the normal MS salt concentration in the inoculation and co-cultivation medium (the latter solidified with 6 g/l agarose) and Agrobacterium inoculum density at 600 nm of 1 - 4 proved to be most efficient. Impact of vector/strain combination on transformation efficiency “Super-virulent” Ti plasmids/strains have been considered as one of the major reasons for recent success in achieving Agrobacterium-mediated transformation of cereals (Hiei et al., 1994; Ishida et al., 1996; Tingay et al., 1997; Zhang et al., 1997). Two strategies based on the “super-virulent” Ti plasmid - pTiBo542 have been developed. The first one is based on strain EHA101 which is derived from the wild-type strain C58, but carries pTiBo542 (Hood et al., 1986). The second strategy involves what is known as a “super-binary” vector in which a DNA fragment (vir G and virB) from the virulence region of pTiBo542 is introduced into a small, T-DNA carrying plasmid (Komari et al., 1990) used in a binary vector system (Hoekama et al., 1983; An et al., 1988). In our experiments we employed both strategies. A range of plasmids was used to establish the impact of vector “virulence”. The plasmids pAL155, pAL156, PAL157 and pSoup were provided by Dr D Lonsdale of The John Innes Centre. The difference between pAL155 and pSoup is in the presence/absence of vir G542 deriving from pTiBo542. Plasmids pAL156 and pAL157 contain the gusA gene with an intron inserted at nt 385, and the bar gene, both genes driven by the maize Ubiquitin1 promoter plus intron; pAL157 have an additional vir G542 copy in the vector backbone. The plasmid pB4Th (Figure 3) created in The Norman Borlaug Institute by the use of the bacteriophage-derived Cre/loxP site-specific recombinase system was also used in this experiment. Having three marker genes nptII, bar and hph within two independent T-DNA regions it provides a means for not only comparing selection efficacy of these selectable markers, but allows also their segregational removal, responding to the necessity of development of “clean-gene” technologies. Hence, once its optimal Agrobacterium host strain has been established, this vector could be used to evaluate the impact of the marker gene on regeneration after transformation. The pB4Th contains the gusA gene with intron 2 from the ST-LS1 gene of potato driven by 35S CaMV promoter, thus giving an opportunity to assess the efficacy of the promoter for detection of transient gene expression within the barley cell.

Figure 3. Map of pB4.Th.

Project title


MAFF project code

CEO 159

10

Tables 4 and 5 list the “virulence” features of the plasmids and strains used. Table 4. Agrobacterium tumefaciences strains and their “virulence” background

Strain “Virulence” plasmid LBA4404 None C58 None EHA101 pTiBo542 AGL1 pTiBo542

Table 5. Plasmids and virG gene derived from pTiBo542

Plasmid Copy number of virG gene

pB4.Th 0 pSoup 0 pAL155 1 pAL156 0 pAL157 1

On MS3 medium for inoculation/co-cultivation the plasmid combinations: pSoup + PAL157, pSoup + pAL157, pAL155 + pAL156 and pAL155 + pAL157 gave markedly higher transformation frequencies when they were harboured in AGL1 than in LBA4404 (Table 6). Apparently this difference comes from the different virulence background of these strains. Presence of an additional virG 542 gene copy gives an extra positive impact on transformation efficiency. Addition of one more virG gene, however, does not lead to further improvement (in the case of AGL1) or may even have a pronounced negative effect (in the case of LBA4404). Comparison between the transformation efficiencies of combinations pSoup + pAL157 and pAL155 +pAL156 shows that although in both cases there is one copy of virG 542 , its position in trans is more effective than in cis. Table 6. Transient gusA expression in barley (cv. Golden Promise). Freshly isolated IEs were inoculated with A. tumefaciens AGL1 or LBA4404 carrying different combinations of plasmids, resuspended in MS3 medium (OD600 = 1.5) for 30 min and then co-cultivated on MS3 solid medium for 3 days before GUS staining. Values shown are the means + standard deviations of three independent experiments.

Strain Plasmid combination IEs with GUS foci (%)

Av. GUS foci per IE

LBA4404 PSoup + pAL156 29 + 2.3 1.8 + 0.3 LBA4404 PSoup + pAL157 13 + 1.4 0.6 + 0.1 LBA4404 pAL155 + pAL156 48 + 4.2 2.7 + 0.3 LBA4404 pAL155 + pAL157 18 + 2.1 1.2 + 0.1 AGL1 pSoup + pAL156 87 + 2.6 6.0 + 0.9 AGL1 pSoup + pAL157 82 + 2.1 5.3 + 0.5 AGL1 pAL155 + pAL156 95 + 1.7 9.6 + 1.2 AGL1 pAL155 + pAL157 92 + 1.2 8.1 + 1.3

Further improvement of the transformation efficiency was achieved by using RMS3, instead of MS3 medium (Table 7). Again the combination of “super-virilent” strain and “ordinary” plasmid was much more successful than that of “ordinary” strain and “ordinary plasmid” (EHA101/pB4.Th vs C58/pB4.Th) or “ordinary” strain and “super-virulent” vector (LBA4404/pSoup + pAL157 vs AGL1/pSoup + pAL156) . However the result from the combination of AGL1/pB4.Th is difficult to explain on the basis of the strain’s virulence background characteristics.

Project title


MAFF project code

CEO 159

11

Plasmid pB4.Th was much less effective than pSoup + pAL156 although neither of them carries any additional virG gene. However, the gusA gene in pB4.Th is driven by a CaMV 35S promoter whereas in pAL156 it is under control of the maize Ubiquitin promoter. This might be the main reason for the observed difference as the latter promoter is far more effective in a monocotyledon “environment” than the former one (Christensen et al., 1992). Table 7. Transient gusA expression in barley IEs transformed under optimized conditions with different A. tumefaciens strains carrying different combinations of plasmids. Freshly isolated IEs were inoculated with bacterial suspensions in RMS3 medium (OD600 = 1.5) for 2 h and then co-cultivated on RMS3 solid medium for 4 days before GUS staining. Values shown are the means + standard deviations of three independent experiments.

Strain Plasmid combination

IEs with GUS foci (%)

Av. GUS foci per IE

GUS foci distribution (scutellum/elsewhere)

AGL1 pSoup + pAL156 100 + 0.0 80 + 8.6 45/35 (56 + 6.7%) AGL1 pSoup + pAL157 100 + 0.0 84 + 7.4 50/34 (60 + 5.5%) AGL1 pAL155 + pAL156 100 + 0.0 111 + 14.0 69/42 (62 + 5.5%) AGL1 pB4.Th 20 + 2.5 0.2 + 0.0 0/0.2 (0 + 0.0%) EHA101 pB4.Th 50 + 2.9 5.5 + 0.4 1.2/4.3 (11 + 2.4%) C58 pB4.Th 25 + 2.6 1.1 + 0.2 0/ 1.1 (0 + 0.0%)

Expression of Agrobacterium virulence genes is regulated by a two-component system. The sensor, VirA protein the (virA gene is constitutively expressed) detects small phenolic compounds (e.g. acetosyringone) and then phosphorylates the VirG protein (also produced constitutively), which in its turn activates the transcription of the rest of the vir genes (Lee et al., 1995; Zupan and Zambryski, 1997). The presence and expression of any additional virG gene would produce higher level of virulence (Jin et al., 1987). Another way is by increasing the concentration of the molecules- inducers such as acetosyringone (AS). Depending on the situation, these two ways may have additive effect or non-additive effect on each other. In our experiments where AGL1 was used and its virG542 might have already provided a high level of virulence background, the supplementation of the inoculation medium with AS and additional copies of virG542 seem to have a non-additive effect. When 100 ?M AS was included all four plasmid combinations, whether they have no additional (pSoup + pAL156), one (pAL155 + pAL156 and pSoup + pAL157), or two additional copies of virG542 (pAL155 + pAL157), gave similar levels of transformation efficiency and comparable with those reported in Table 6. In conclusion, the “virulence” background of the Agrobacterium strain is of more importance for transformation efficiency than the “virulence” of the plasmid. The combination “super-virulent” strain/ “super-binary vector” is the combination of choice. Addition of more than one copy of virG is not necessary. The positioning of the additional copy, however is very important, being more effective in trans. Acetosyringone does not have an impact on transformation efficiency. c) Stable transformation of barley immature embryos or embryogenic cultures Construction of a novel series of binary plasmids for the facile creation of complex T-DNA vectors which incorporates “clean-gene” facilities The challenge of Agrobacterium-mediated transformation and selection of stable transformants from the recalcitrant cereals demands a high degree of vector design-flexibility. To satisfy this requirement the pBECKS2000 series of binary vectors was constructed (Figure 4) in The Norman Borlaug Institute by use of the bacteriophage-derived Cre/loxP mediated recombination system (McCormac et al., 1999). The pBECKS2000 vectors allow the marker genes and target genes intended for co-transfer as a single or two independent T-DNA units, to be ligated into different plasmid entities via independent strategies and co-integration of them is then achieved through a site-specific recombination system. These vectors enable not only rapid generation of complex T-DNA molecules but also provide facilities for removal of transformation markers from transgenic plants via 1) a Cre/loxP facility for site-specific excision of marker genes from the

Project title


MAFF project code

CEO 159

12

plant genome, or 2) co-transfer of target and marker genes as two independent T-DNAs within a single-strain Agrobacterium system, providing the potential for segregational loss of marker genes. Several of these vectors were used in our experiments aimed at stable transformation of barley via Agrobacterium-mediated gene delivery (see below).

Figure 4. Maps of pBECKS2000 series of binary vectors. Stable transformation of barley immature embryos The protocol developed after the evaluation of the parameters influencing T-DNA delivery at an pre- integration state was used. Briefly, freshly isolated IEs of barley were inoculated with Agrobacterium AGL1/ pAL155 + pAL156 (OD600 = 1. 5) in RMS3 medium for 2 h and then co-cultivated on RMS3 medium solidified with 6 g/l agarose for 3 days. Although embryo wounding did not have impact on transient gusA expression, the axis of the embryos was removed before Agrobacterium transformation, as it was proved that without this treatment all immature barley embryos tended to develop roots and shoots instead of callus. After co-cultivation the embryos were transferred to the selection medium - MS3 supplemented with 150 mg/l Timentin and 3 mg/l bialaphos and solidified with 2.5 g/l Phytagel. Alteration of the salt concentration from 0.1 X to the original one (1 X) and the type of gelling agent from agarose to Phytagel was done because they lowered the percentage and produced poor quality bialaphos-resistant calluses lacking any regeneration capacity. Subcultures on selection medium were carried out at intervals of two weeks. Bialaphos-resistant calluses usually emerged after three weeks (Figure 5a). White or white-yellow resistant calluses sometimes regenerated directly from embryo scutellum edges (Figure 5b). Throughout the selection process they retained their colour (white/ white-yellow). However, there was another more frequently observed way of producing bialaphos-resistant calluses - after enlargement during the first week of selection, many embryos produced compact calluses originating from their scutella. Two-three weeks later almost all of these calluses turned yellow or yellow-brown, and white or yellow-white compact calluses began to emerge from some of them (Figure 5c). Similar callus initiation was observed with non-transformed embryos, however at a much lower frequency. Usually these non-transformed calluses soon died. Transformed white or white-yellow calluses cut into small

Project title


MAFF project code

CEO 159

13

pieces and subculured onto fresh selection MS3 medium every two weeks, remained fresh and grew healthily. However, after two months of selection only 10 - 20 % of them produced positive GUS stains (Figure 5d). Extension of the selection period for another three months did not increase the percentage of the GUS positive calluses. The transformation rate was very low - < 0.5%. And although a few plantlets were regenerated, none of them was proved to be positive after Southern hybridisation.

Figure 5. Production of bialaphos-resistant calluses: a – transformed embryos began to produce bialaphos-resistant calluses after 2 -3 weeks selection; b – yellow-white bialaphos-resistant calluses emerged from the edge of scutellum that had undergone selection for about 3 weeks; c – white or yellow-white bialaphos-resistant calluses generated from yellow and yellow-brown old calluses after 4 – 5 weeks selection; d – GUS staining of bialaphos-resistant calluses which survived selection for more than 2 months. These data lead to two main conclusions: 1. bialaphos is not an effective selection agent 2. the long exposure to bialaphos, probably in a combination with 2,4 D dramatically decreases the regeneration capacity of the transformed cells Hence, 1. alternative selection procedures needed to be employed 2. other plant growth regulators should be tried to replace 2,4-D for callus initiation 3. to increase the efficiency of the protocols aiming for stable transformation of barley, explants with more cells having high regeneration capacity needed to be exposed to Agrobacterium inoculation. Stable transformation of cell suspension cultures derived from barley IEs and microspores Cell suspension cultures derived from IEs and microspores of Dissa and Igri were inoculated and co-cultivated with appropriate Agrobacterium strain/vector combinations. Agrobacterium strains/vectors

Project title


MAFF project code

CEO 159

14

EHA101/pBECKS400, LBA4404/pTOK233 or EHA101/pBECKS400 were employed. After two to three 4 week cycles on selection medium with hygromycin (50 mg/l) or PPT (5-10 mg/l) stable transformed calluses, showing uniform GUS staining were produced (Figure 6E). Transformation frequencies of 0.3-3% (based on the numbers of transformants divided by the numbers of inoculated cultures) were obtained (Table 8). However, these cultures were not able to regenerate on shoot induction medium due to the long culture periods.

Figure 6. Analysis of early and stable transgene expression in Agrobacterium co-cultivated barley cell suspension cultured cells. Transient gene expression after co-cultivation with EHA101/pBECKS400red/bar (A,B); or LBA4404/pTOK233 (C), stable gene expression after co-cultivation with LBA4404/pTOK233 after one(D) or two (E) cycles of selection with hygromycin. Table 8. Transformation frequencies of barley cell suspension cultures

Cultivar Number of explants Agrobacterium strain/ve ctor No of transformants (transformation frequencies, % )

Dissa 670

EHA101 pBECKs400.gus/hpt 2 (0.3%) Calluses

Igri 690 LBA4404 pTOK233

20 (2.9%) Calluses

Dissa 680

EHA101 pBECKs400.gus/bar 13 (1.9%) Calluses

Igri 750

EHA101 pBECKs400.gus/bar 3 (0.4%) Calluses

Project title


MAFF project code

CEO 159

15

Stable transformation of barley embryogenic callus Embryogenic callus cultures derived from IEs of cv. Golden Promise and cv. Dissa were used as material to co-cultivate with Agrobacterium strain/vector LBA4404/pTOK233, EHA101/pBECKS2000.5, EHA101/pBECKS2000.7, EHA101/ pB4Th, AGL1/pAL155+156 and EHA105/pMan. Selection was carried out on a modified MS medium as mentioned above. Altogether 5 hygromycin, 6 mannose and 3 bialophos resistant cultures were obtained after 2 rounds of selection with 3-4 weeks cycles (Table 9). Some of these transformed cultures regenerated shoots or plantlets on plant regeneration medium. These putative transformants will be further characterised and used to produce seeds for transgene inheritance analysis.

Table 9. Transformation of barley embryogenic callus cultures Cultivar Explant

number Agrobacterium strain No of transformants

Dissa 150 EHA101 pBECKs400.gus/bar 3 Plantlets Golden Promise 120 EHA101 /pBECKs2000.5 .gfp/bar 3 Regenerants resistant to bialaphos Dissa 250 LBA4404 /pTOK233 3 Calluses resistant to hygromycin Dissa 75 EHA105 /pMAN.man 4 Regenerants resistant to mannose Golden Promise 75 LBA4404 /pTOK233, EHA105/ pMAN.man(1) 2 Calluses resistant to mannose Dissa 95 EHA101 /pBECKS2000.7.gfp/hyg 2 Calluses resistant to hygromycin Golden Promise 140 EHA101/pB4Th Cultures in hygromycin or bialophos selection Golden Promise 125 AGL1/pAL155+156 Cultures in bialaphos selection

(1) Co-transformation with two strain/plasmid systems

By using fresh embryogenic callus cultures derived from microspores of Dissa, and PPT as selective agent, 3 putative transformed plants were producted - a transformation frequency of 2% (Table 9). However, these plants were not successfully established in soil and did not set seeds so genetic analyses could not be carried out.

d) Molecular analysis of the transformed calli and transgenic plants Southern blot hybridisation according to the standard protocols was carried out with some of the transformed cell lines. Genomic DNA was isolated from calli that have been transformed with EHA101/pBECKs400.gus/hpt, EHA101/pBECKS400.gus/bar or LBA4404/pTOK233 and used to hybridize to the hph or bar and gusA gene as a probe (Figures 7 and 8). Endonuclease digestion was carried out with enzymes which released an internal fragment of the transgene being probed and which should, therefore, produce a common pattern of hybridisation. In accord with this, genomic DNA from the hygromycin resistant barley lines A56-P-1 and A56-P-2, which had been digested with EcoRI or BamHI, produced identical patterns of hph-hybridising bands (Figure7a, b [E, B]) and these corresponded with the sizes predicted by restriction analyses of the co-cultivated LBA4404/pTOK233 strain (Figure 7c [E, B]). Likewise, hybridisation of the gusA probe to HindIII or EcoRI-restricted DNA revealed common single bands (Figure 7d, e [H,E]) and these also corresponded with the co-cultivated Agrobacterium (Figure 7f). In the case of barley line A49-ch-2, hybridisation of the hph probe to DNA restricted with HindIII, EcoRI, Xbal or BamHI revealed single bands which corresponded with those obtained in restriction digests of the co-cultivated strain EHA 101 (Figure 7g, I[H, E, X, B]), as did hybridisation of the gusA probe to HindIII, KpnI,Xbal or BamHI-restricted DNA (Figure 7j, l[H, K, X, B]). The consistency of these predicted bands indicates that rearragements or deletions had not occurred within the intervening portion of the T-DNA in the barley cells.

Project title


MAFF project code

CEO 159

16

Figure 7. Southern blot analysis of hygromycin-resistant callus lines. Callus lines A56-P-1 (a, d) and A56-P-2 (b, e) were obtained following the co-cultivation of cell suspension cultured cells with LBA4404 pTOK233. Callus line A-49-ch-2 (g, j) was obtained following the co-cultivation of CSC with EHA101 pBECKs400.gus/hpt. Genomic DNA was also extracted from a no-transformed callus line (h, k). Total genomic DNA was digested with HindIII (H), XbaI (X), EcoRI (E), BamHI (B), KpnI (K) or left intact (U), followed by fraction by electrophoresis on a 0.6 % agarose gel and transfer to nylon membrane. Blots were allowed to hybridize with 32P-dCTP-labled probes for the hph gene (a, b, g, h) or gusA (d, e, j, k). Panels (c, f) show, respectively, the size of hph and gusA-hybridizing bands which correspond to the co-cultivated Agrobacterium LBA4404 pTOK233. Panels (i, l) show, respectively the size of hph and gusA-hybridizing bands which correspond to the cocultivated Agrobacterium EHA101 pBECKs400.gus/hpt. If DNA is resticted with an enzyme which recognizes a single site within the T-DNA then the hybridisation pattern obtained will depend on the presence of a second site within the surrounding DNA; hence this pattern should be unique for each independent line. In accord with this, barley lines A56-P-1 and A56-P-2 contained hph-hybridising bands in the XbaI or HindIII restricted DNA which were distinct for each line (Figure 7a, b [H, X]) and also different from the those of the co-cultivated Agrobacterium (Figure 7c, (H, X). The number of bands in each of these barley lines indicated 1 and 2 – 3 copies, respectively, of the hph gene (Fig 7a, b (H, X). Likewise, hph-hybridisation to KpnI-restricted DNA from barley line A49-ch-2 indicated 2 T-DNA copies (Figure7g [K]), each with a mobility distinct from that of the co-cultivated EHA101 plasmid (Figure 7i [K]).

Project title


MAFF project code

CEO 159

17

When genomic DNA from a non-transformed barley callus line was allowed to hybridise to the hph and gusA probes, some non-specific hybridisation to the unrestricted samples was revealed (Figure 7h, [U]). But no signal could be detected within the digested control samples (Figure 7h, k [H, E, B]), thus confirming that the banding patterns of the transformed lines were specific to the T-DNA genes. PPT-resistant barley callus line A47-2-6 and PPT-resistant plantlet A87-1-b also demonstrated banding pattern identical to that of the co-cultivated Agrobacterium, when digestion was performed to release the bar gene (Fig. 8a, d, e (H,) or gusA gene (Fig. 8f, i, j (H) from the T-DNA. In contrast, digestion with enzymes which linearised the gene within the T-DNA produced hybridisation patterns to the bar (Fig. 8a, d, e (Bg) and gusA (Fig. 8f, i, j (Bg) probes that differed from that of the co-cultivated Agrobacterium.

Figure 8. Southern blot analysis of a PPT-resistant callus line A47-2-6 (a, f) and a PPT-resistant plantlet A87-1-b (d, i) with probes for the T-DNA-contained bar (a-e) or gusA (f-j) genes. Control samples are shown in panels b, c, g and h; predicted Agrobacterium bands are shown in panels e and j. Callus line and plant were obtained following the co-cultivation of CSC and embryogenic callus with EHA101/pBECKS400.gus/bar. Genomic DNA was also extracted from a non-transformed callus line (b, c, g, h). Total genomic DNA was digested with HindIII (H) and BglII (Bg), followed by electrophoresis on a 0.6% agarose gel and transfer to nylon membane.

CONCLUSIONS AND FUTURE DEVELOPMENT Efficient procedures for regeneration of barley plants from in vitro cultures of immature embryos and microspores have been established and procedures for efficient transgene delivery via Agrobacterium-mediated transformation have been optimised. However, attempts based on these optimised procedures, to recover transgenic plants by direct use of immature embryos for co-cultivation with Agrobacterium, failed. The transformed cells completely lost their regeneration potential due to the long periods of selection pressure, coming mainly from the fact that the selective agent used – bialaphos – was not effective. That is why in our further work other types of explants were used for co-cultivation together with other selective agents. By using fresh embryogenic callus cultures derived from microspores of barley cultivar Dissa and PPT as selective agent, 3 trangenic plants were recovered. This protocol (Figure 9) delivered a transformation efficiency of 2%.

Project title


MAFF project code

CEO 159

18

Figure 9

Isolation of microspores at mid-late uninucleate stage ?

Induction of embryogenic callus on modified N6 medium (Wu et al., 1998) suppl emented with 0.5 mg/l 2,4-D plus 0.5 mg/l kinetin

? Cocultivation with Agrobacterium EHA101 pBECKS 2000 (OD600 value of about 1.0 ) for 2-4 days at 25 ºC

? Disinfection with cefotaxime (400 mg/l) and culture on modified MS medium (Cho et al., 1998) containing cefotaxime for 1-2

weeks ?

Callus selection for 2 X 3-4 week cycles on callus induction medium (Cho et al., 1998) containing PPT (5 mg/l) ?

Plant regeneration selection on FHG medium (Hunter, 1998) containing PPT (5 mg/l) ?

PCR and Southern blot analysis of putative transformants ?

Genetic analysis of progeny of the transformants Although Tingay et al. first reported successful transformation of barley cv. Golden Promise IEs by cocultivating with Agrobacterium in 1997, few researchers have been able to repeat the experiments. Recently, an improved procedure based on hygromycin selection and a more virulent Agrobacterium strain AGLO was described (Wang et al., 2000) but it is still difficult to transform other barley cultivars by Agrobacterium cocultivation. Our experiments indicated that barley cultivars Dissa and Igri were amenable to transformation, via our improved inoculation and co-cultivation procedures. The loss of key personnel has slowed progress but large numbers of plantlets, regenerated from embryogenic callus derived from IEs are now growing on selection media containing hygromycin. There are indications of high transformation efficiencies. The new protocol is presented in Figure 10. Figure 10

Isolation of immature embryos 14 days after anthesis ?

Induction of embryogenic callus on modified MS medium (Cho et al., 1998) supplemented with 2.5 mg/l dicamba ?

Cocultivation with Agrobacterium EHA101 pBECKS 2000 (OD600 ?1.0 ) for 2-4 days at 25 ºC ?

Disinfection with cefotaxime (400 mg/l) and culture on modified MS medium (Cho et al., 1998) containing cefotaxime for 1-2 weeks

? Callus selection for 2 X 3-4 week cycles on callus induction medium (Cho et al., 1998) containing hygromycin (50 mg/l)

? Plant regeneration/selection on FHG me dium containing hygromycin (25 mg/l)

? PCR and Southern blot analysis of putative transformants

? Genetic analysis of progeny of the transformants

It should be noted that when embryogenic callus derived from barley cv. Golden Promise was used for co-cultivation with Agrobacterium EHA105/pMan 2 calli growing on mannose-containing medium. A

Project title


MAFF project code

CEO 159

19

transformation frequency of 2.7% (based on the number of transformed cultures divided by the number of inoculated calluses) was indicated. Although this experiment is still at early stage there are indications that mannose-based selection procedures could be very effective for Agrobacterium-mediated barley transformation. Trifonova et al. (2001) recently described a range of procedures for Agrobacterium-mediated transformation of barley immature embryos. We are beginning a comparative analysis of their system with our own. Future development of this project (at the Norman Borlaug Institute’s own expense) will include comparison of the selectable marker genes used for their efficiency for transgenic plant production, large-scale molecular characterisation of transformation events, progeny analysis for selectable marker- free transgenic plants and deployment of agronomic or industrially useful genes for crop improvement. The results will, of course, be reported to the DEFRA. References 1. An GH, Evert PR, Mitra A., Ha SB (1988) Binary Vectors. In: Plant Molecular Biology manual A3 (Gelwin SB & Schilperoort

RA eds). Dodrecht: Kluwer Academic Press, pp 1-19. 2. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Dunkan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat

mediated by Agrobacterium tumefaciens. Plant Physiol. 115:971-980. 3. Chibbar RN, Kartha KK, Datla RSS, Leung N, Caswell K, Mallard CS, Steinhauer L (1993). The effect of different promoter

sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells. Plant Cell Rep. 12:506-509. 4. Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFR as a vital reporter in plants. Current

Biotechnol. 6:326-330. 5. Christensen AH, Sharrock RA,Quail PH (1992) Maize polyubiquitin genes - structure, thermal perturbation of expression and

transcript splicing, and promoter activity following transfer to protoplasts by lectroporation. Plant Mol. Biol. 18:675-689. 6. Cho MJ, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars trough improvement of regenerability and

decreased albinism. Plant Sci. 138:229-244. 7. Corneyo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic

rice. Plant Mol. Biol, 23:567-581. 8. Jin S, Komari T, Gordon MP, Nestler EW (1987) Genes responsible for the supervirulence phenotype of Agrobacterium

tumefaciens A281. J. Bacteriol. 169:4417-4425. 9. Hiei Y, Ohta S, Komari T, Kumashiro S (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium

and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271-282. 10. Hoekama A, Hirsch PR, Hooykaas PJ, Schliperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-

region of the Agrobacterium tumefaciens Ti-plasmid. Nature, 303:179-180. 11. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a

region of the pTiBo542 outside of T-DNA. J. Bacteriol. 168:1291-1301. 12. Hunter CP (1988) PhD Thesis, Wye College, University of London 13. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996). High efficiency transformation of maize (Zea mays L.)

mediated by Agrobacterium tumefaciens. Nat. Biotechnol.14:745-750. 14. Komari T (1990) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from

the virulence region of pTiBo542. Plant Cell Rep. 9:303-306. 15. Lee YW, Jin S, Sim WS, Nester EW (1995) Genetic evidence for direct sensing of phenolic compounds by the VirA protein of

Agrobacteriium tumefaciens. Proc. Natl. Acad Sci. USA 92:12245-12249. 16. Li L., Qu R, De Kochko A, Faquet C, Beachy RN (1992) An improved rice transformation system using the biolistic method.

Plant Cell Rep. 12:250-255. 17. McCormac AC, Wu H, Bao M, Wang Y, Xu R, Elliott M, Chen D (1998) The use of visual marker genes as cell-specific

reporters of Agrobacterium-mediated T_DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) Euphyica, 99:17-25.

18. McCormac AC, Elliott, MC, Chen DF (1999) pBCKS2000: a novel plasmid series for the facile creation of comlex binary vectors, which incorporates “clean-gene” facilities. Mol. Gen. Genet. 261:226-235.

19. Murashige T and Skoog F (1962) A evised medium for apid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15:473-497.

20. Pang S, DeBoer DL, Wan Y, Ye G, Layton JC, Neher M, Armstrong C, Fry JE, Hinchee MAW, Fromm ME (1996) An improved green fluorescence protein gene as a vital marker in plants. Plant Physiol. 112:893-900.

Project title


MAFF project code

CEO 159

20

21. Quattrochio F, Wing JF, Leppen H, Mol JN, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5: 1497-1512.

22. Taylor MG, Vasil V, Vasil I (1993) Enhanced GUS gene expression in cereal/grass cell suspensions and immature embryos using the maize ubiquitin based plasmid pAHC25. Plant Cell Rep. 12:491-495.

23. Tingay S, McElroy D, Kala R, Fieg S, Wang M, Thornton S, Brettel R (1997) Agrobacterium tumefaciens mediated barley transformation. Plant J. 11:1369-1376.

24. Trifonova A, Madsen S, Olsen A. (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Science 161: 871-880.

25. Wan Y and Lemaux PG (1994) Generation of large number of independently transformed fertile barley plants. Plant Physiol. 104:37-48.

26. Weeks TJ, Anderson OK, Blechl AE (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol. 102:1077-1084.

27. Wu H, McCormac AC, Elliott MC, Chen DF (1998) Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare). Plant Cell Tissue and Organ Cult. 54:161-171.

28. Zhang J, Xu R, Elliott MC, Chen D (1997) Agrobacterium mediated transformation of elite Japonica and Indica rice varieties. Mol. Biotechnol. 8:223-231.

29. Zupan JR and Zambryski PC (1997) The Agrobacterium DNA transfer complex. Critical Rew. Plant. Sci, 16(3):279-295. PUBLICATIONS AND OTHER OUTPUTS 1.0 The pBECKS 400 and pBECKS 2000 A. tumefaciens vectors are now available for use. 2.0 Publications in refereed journals: McCormac AC, Elliott MC & Chen DF. (1998) A Simple Method for the Production of Highly Competent Cells of Agrobacterium for transformation via Electroporation. Molecular Biotechnology, 9, 155-195. McCormac A, Wu HX, Bao MZ, Wang YB, Elliott MC & Chen DF. (1998) The Use of Visual Marker Genes as Cell-Specific Reporters of Agrobacterium-mediated T-DNA Delivery to Wheat (Triticum aestivum L.) and Barley (Hordeum vulgare L.). Euphytica, 99, 17-25. Wu H-X, McCormac AC, Elliott MC & Chen DF. (1998) Agrobacterium-mediated Stable Transformation of Cell Suspension Cultures of Barley (Hordeum vulgare, L.). Plant Cell Tissue and Organ Culture, 54, 161-171. Wu H, McCormac AC, Elliott MC & Chen D-F. (1999) Agrobacterium-mediated Stable Transformation of Barley. In: Plant Biotechnology and In Vitro Biology in the 21st Century (A Altman, M Ziv & S Izhar, Eds). Kluwer Academic Publishers, pp 231-234. McCormac AC, Elliott MC & Chen DF. (1999) pBECKS 2000: A Novel Plasmid Series for the Facile Creation of Complex Binary Vectors which Incorporates Clean-gene Facilities. Molecular and General Genetics, 261, 226-235. McCormac AC, Fowler MR, Chen D-F & Elliott MC. (2001) Co-transformation with Independent T-DNAs for Segregational Separation: the Influence of Vector Design. Transgenic Research, 10, 143-155. Ke X-Y, McCormac AC, Harvey A, Lonsdale D, Chen D-F & Elliott MC. (2001) Manipulation of Discriminatory T-DNA Delivery into Cells of Immature Embryos of Barley and Wheat by Agrobacterium. Submitted to Journal of Experimental Botany.

Project title


MAFF project code

CEO 159

21

3.0 Conference presentations: Chen D-F, Ke X, McCormac A, Sorokin A, Wu H & Elliott MC. (1998) Tissue Electroporation and Agrobacterium-mediated Transformation of Wheat and Barley. MAFF Crop Transformation Workshop, IACR Rothamsted, July 2-3, 1998. Elliott MC. (1998) Plant Biotechnology: the Way to Sustainable Agriculture? (Plenary Lecture). Institute of Experimental and Technological Biology (IBET) Workshop on Plant Biotechnology, Oeiras, Portugal, 17-18 September 1998. Elliott MC. (1998) The Role of Gene Manipulation in Crop Improvement. (Plenary Lecture). Greek Ministry of Agriculture Conference, “Genetically Manipulated Organisms in Agriculture”, Athens, Greece. 8/9 December 1998, p.11. Elliott MC, Atanassov A, Bennett J, Chen D-F, Fowler M, Kamínek M, Khush GS, Russinova E, McCormac AC, Scott NW & Slater A. (1998) Towards Sustainable Agriculture via Gene Manipulation. (G Haberlandt Plenary Lecture). Plant Biotechnology and In Vitro Biology in the 21st Century. IX International Congress on Plant Tissue and Cell Culture. Jerusalem, Israel, June 14-19, 1998, p 1. Wu H-X, McCormac AC, Elliott MC & Chen DF. (1998) Agrobacterium-mediated Stable Transformation of Microspore-derived and Immature Embryo-derived Cell Suspension Cultures of Barley (Hordeum vulgare L.). Plant Biotechnology and In Vitro Biology in the 21st Century. IX International Congress on Plant Tissue and Cell Culture, Jerusalem, Israel, June 14-19, p 35. Wu H, McCormac AC, Ke XY, Elliott MC & Chen DF. (1998) Agrobacterium-mediated Stable Transformation of Barley (Hordeum vulgare L.). The Seventh Annual Conference of Life Sciences. Cambridge, UK. 08-6, p 59-60. Elliott MC, Bennett J, Borlaug NE, Chen D-F, Daskalova S, Fowler MR, Kaminek M, Khush GS, McCormac AC, Mishra MK, Peng SB, Rubia L, Scott NW, Sorokin A & Slater A. (1999) Crop Improvement Strategies Must Exploit Gene Manipulation Techniques. (Plenary Lecture). Proceedings of the International Symposium “Auxins and Cytokinins in Plant Development”, Prague, Czech Republic, 26-30 July 1999. Biologia Plantarum 42, S83. Elliott MC, Bennett J, Borlaug NE, Chen D-F, Daskalova S, Fowler MR, Kamínek M, Khush GS, McCormac AC, Mishra MK, Peng SB, Rubia L, Scott NW, Sorokin A & Slater A. (1999) Crop Gene Manipulation is Under Siege. (Plenary Lecture). International Symposium on Plant Genetic Engineering -Towards the Third Millennium, Havana, Cuba, 6-10 December 1999, p 230. Elliott MC, Ke X-Y, Wu H-X, McCormac AC & Chen D-F. (1999) Development of a Routine System for Agrobacterium-mediated Transformation of Barley. MAFF Crop Molecular Genetics Programme Workshop. Cambridge. 26-27 May. Elliott MC. (2000) Let Them Eat Cake – Crop Gene Manipulation and Food Security in the 21st Century. University of Calgary Research Seminar, Calgary, Canada, 30 June 2000. Elliott MC, Atanassov AI, Bennett J, Borlaug NE, Chen D-F, Chen Z-L, Daskalova S, Devi S, Dowswell C, Fowler MR, Kamínek M, Khush GS, McCormac AC, Mishra MK, Peng SB, Rubia L, Scott NW, Sorokin A,

Project title


MAFF project code

CEO 159

22

Xu Z-H & Slater A. (2000) GM Crops - Why is There a Crisis? (Plenary Lecture). ICGEB Global Consortium on Plant Biotechnology and Genomics; Sofia, Bulgaria, 16-19 May 2000, p2. Elliott MC, Atanassov AI, Bennett J, Borlaug NE, Daskalova S, Devi S, Dowswell C, Fowler MR, Kaminek M, Ke X-Y, Khush GS, McCormac A, Mishra MK, Peng S, Rubia L, Scott NW & Slater A. (2000) Transgenic Crops – A Rational Analysis. (Plenary Lecture). Proceedings of “The Future of Transgenic Crops in Romanian Agriculture”, Bucharest, 6-8 November 2000, p 2. Elliott MC, Atanassov AI, Bennett J, Borlaug NE, Daskalova S, Fowler MR, Kamínek M, Ke X-Y, Khush GS, McCormac AC, Mishra MK, Peng SB, Rubia L, Scott NW, Sorokin A & Slater A. (2000) Crop Improvement Strategies Must Exploit In Vitro Techniques. (Plenary Lecture). EUCARPIA XVIII International Conference on Maize and Sorghum Genetics and Breeding; Belgrade, Yugoslavia, 4-9 June 2000, p 48. Elliott MC, Atanassov AI, Bennett J, Borlaug NE, Daskalova S, Fowler MR, Kaminek M, Ke X-Y, Khush GS, Peng S, Scott NW, Slater A & Xu Z-H. (2000) Plant Molecular Biology – Applications in Developing Countries (Plenary Lecture), VI International Congress of Plant Molecular Biology; Quebec, Canada, 18-24 June 2000, SO3-35. Elliott MC. (2001) Plant Gene Manipulation – Towards the Blue Horizon (Plenary Lecture). Proceedings of the ICRO-UNESCO Workshop “Molecular and Cellular Aspects of Gene Transfer in Plants”, Sofia, Bulgaria, 7-19 May 2001. Elliott MC, Bennett J, Borlaug NE, Daskalova S, Fowler MR, Khush GS, Peng S, Rubia L, Scott NW, Sorokin A, Slater A & Zhang CL. (2001) Crop Gene Manipulation: the Way to Sustainable Agriculture and World Food Security? (Plenary Lecture). XVII International Conference on Plant Growth Substances, Brno, Czech Republic, 1-6 July 2001, p 45. Elliott MC, Bennett J, Borlaug NE, Daskalova S, Fowler MR, Khush GS, Peng S, Rubia L, Scott NW, Slater A & Zhang CL. (2001) Genetically Enhanced Crops: Sustainable Agriculture and Biodiversity (Plenary Lecture). Russian Academy of Sciences Workshop, “Ecological Aspects of Plant Genetic Engineering”, Moscow, 27-28 September 2001. Please press enter

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